Lab 4: Building a Better World (AKA Fluvastatin Synthesis - Reduction of FLEXAT to DIOLAT)

Transcription

1 Life Sciences 1a Laboratory all 2006 Lab 4: Building a Better World (AKA luvastatin Synthesis - Reduction of LEXAT to DIOLAT) Goals of the lab: 1. To synthesize racemic Diolat from lexat 2. To observe the progression of a chemical reaction by TLC. To employ safe synthetic organic chemistry lab techniques Introduction LEXAT and DIOLAT are two chemical intermediates in the synthesis of the cholesterol lowering drug fluvastatin sodium (sold under the name Lescol XL by ovartis). In this experiment, you will reduce the hydroxyketone LEXAT to the diol DIOLAT using sodium borohydride and monitor the reaction by thin layer chromatography. The DIOLAT product from this reaction will be composed of both syn and anti diastereomers. How the diastereoselectivity of this reaction is controlled in the industrial synthesis will be discussed. OH O O O O LEXAT DIOLAT O a Lescol XL (fluvastatin sodium) References This procedure was modified by Dr. Allen Aloise from the industrial synthesis of fluvastatin sodium by Sandoz Inc., a ovartis company. The LEXAT used in this experiment was generously donated by ovartis. 7

2 Life Sciences 1a Laboratory all 2006 Reading Visit for more information about Lescol XL. Introduction The development of hydride reagents for the reduction of carbonyl derivatives has been of great significance in synthetic organic chemistry. The most common commercially available metal hydrides are sodium borohydride (abh 4 ) and lithium aluminum hydride (LiAlH 4 ). Both hydride reagents react with a broad range of electrophilic substrates with the transfer of a hydride (H-) ion. The general stoichiometry followed in the reduction of ketones and aldehydes is illustrated below for abh 4. The liberation of the desired alcohol is usually accomplished by the subsequent addition of water, acid, or aqueous hydrogen peroxide (in the case of borohydride reageants). 4 R O R ' abh 4 H O B a + aq. H 2 O H OH 2 4 R R' R R' 4 The hydride donor capabilities of sodium borohydride and lithium aluminum hydride are illustrated below, with the carbonyl substrates arranged in decreasing ease of reduction. Lithium aluminum hydride is an exceedingly powerful reducing agent and is capable of reducing even less reactive functional groups than those shown. On the other hand, sodium borohydride is an exceptionally mild reducing agent, only strong enough to reduce the most reactive of carbonyl functional groups: aldehydes, ketones, and acid chlorides. O R H aldehyde R OH abh 4 LiAlH 4 (yes) (yes) O R ketone R ' R OH R' (yes) (yes) O R OR' ester R OH (no) (yes) You can see why sodium borohydride is the appropriate choice for our reduction of interest. We wish to reduce only the ketone in the LEXAT starting material and leave the aromatic rings, double bond, and ester intact. Sodium 74

3 Life Sciences 1a Laboratory all 2006 borohydride is only powerful enough to reduce the ketone functionality. You will be carrying out the reaction below in this experiment. OH O O O 1. abh 4 TH, 0 C 2. H 2 O 2 O LEXAT DIOLAT The LEXAT starting material we will be using is drawn with a squiggly line connecting the carbon and oxygen atom of the alcohol functionality. This is to indicate that our sample is a mixture of R and S stereocenters at the alcohol carbon. Because LEXAT contains only a single stereogenic center, and an equal number of R and S enantiomers are present, the mixture is said to be racemic. An alternative way of showing this would be to draw out each of the two enantiomers: S OH O O S-LEXAT O + R OH O O R-LEXAT O racemic mixture When our sodium borohydride reduction is carried out, DIOLAT is formed as a mixture of four diastereomers. If you look carefully, you can see that the two possible syn-diol arrangements are enantiomeric and that the two possible antidiol arrangements are enantiomeric as well. The product of our reaction will be a racemic mixture of syn and anti products. 75

4 Life Sciences 1a Laboratory all 2006 S R syn-diolat O S S anti-diolat O + R S O R R O syn-diolat anti-diolat syn-enantiomers anti-enantiomers The formation of syn-diolat is required in the industrial synthesis of fluvastatin sodium. Chemists were able to achieve this result by employing diethylmethoxyborane (DMB) as an additive in the reaction. DMB forms a sixmember ring complex with the alcohol and ketone, resulting in a directed hydride addition to afford predominantly the syn-diolat product. 1. abh 4 OH O O O (C 2 H 5 ) 2 BO TH, -78 C O LEXAT ±syn-diolat 76

5 Life Sciences 1a Laboratory all 2006 Procedure OH O O O 1. abh 4 TH, 0 C O LEXAT DIOLAT LEXAT abh 4 TH H 2 O 2 DIOLAT (0%) MW (g/mol) mp ( C) bp ( C) density (g/ml) mg ml mmol equiv Add 1 ml of tetrahydrofuran (TH) to a glass vial with a magnetic stir bar at the bottom. 2. Cool the vial with TH to 0 C by clamping the reaction vessel to the vertical metal bar at your bench and partially submerging it in an ice water bath resting on a magnetic stirrer.. Turn the magnetic stirrer on and make slight adjustments to your set-up so that the stir bar is spinning in a consistent and controlled manner. 77

6 Life Sciences 1a Laboratory all 2006 igure. Assembled reaction vial apparatus 4. Go to the balance area and obtain a piece of weighing paper. old the weighing paper in half and then open it up so that a deep crease runs across it. Place the weighing paper on a digital balance and press the tare button. This will reset the balance to zero. Using a spatula, carefully transfer 6. mg of sodium borohydride from the reagent bottle to the weighing paper. a. This is a small amount of material, therefore you may need to close the side doors of the balance to keep atmospheric disruptions from making the balance readings jump around. 5. Recap the reagent bottle when finished and carefully pick up the weighing paper and return to your bench with it. 6. Unscrew the top of your reaction vessel and use the weighing paper as a funnel to add the sodium borohydide to the TH. a. You may need to use your spatula to scrape off all of your reagent from the weighing paper. 78

7 Life Sciences 1a Laboratory all 2006 igure. The weigh station. 7. Repeat the above procedure to weigh out 100 mg of LEXAT on a piece of weighing paper. 8. Carefully add it to your 0 C solution in portions over 60 seconds. a. The low temperature of the solvent and slow addition of your starting material is to ensure that any heat or bubbles generated from chemical reactions do not boil or splatter your reaction mixture. 9. Cap your reaction vessel and allow your reaction mixture to stir at 0 C for ~ 5 minutes and then remove the ice water bath and allow the reaction to warm to room temperature while stirring. Make sure you record color changes and any other observations you make. 10. Perform a TLC analysis of your reaction, preparing your TLC plate as diagramed below. a. SPOT VERY LIGHTLY. The solutions are very concentrated and if you spot heavily you will mostly see large smears rather than dots. b. Use one of the pre-prepared lexat standards for your starting material spot. This will serve as a standard of comparison. c. The center lane of your TLC plate should receive a spot of both the starting material solution and the reaction mixture. This is your cospot lane and will reveal if there is anything in your reaction mixture that does not correspond to your starting material. You should see two or more spots in this lane if you have created new chemical compounds in your reaction. 79

8 Life Sciences 1a Laboratory all 2006 d. inally, spot the reaction mixture in the right lane of your TLC plate. You will know when your reaction is complete when you no longer see any starting material in this lane. e. Run your TLC plate in a beaker with 5 ml of 40% Ethyl Acetate/60% Hexane. f. Make sure not to let the solvent front reach the top of the plate! time = 1 min time = 5 min time = 15 min 1. develop 2. visualize starting material cospot reaction rxn has only slightly progressed rxn is almost complete rxn is complete 11. The reduction in this experiment is a rapid one and should be complete or nearly so by the time you perform your TLC analysis. Consult with your T if the reaction is not almost completely finished within 20 minutes. Work-Up You will quench your reaction and the remaining unreacted sodium borohydride by adding it to a solution of sodium bicarbonate (ahco ). 12. Obtain a fresh glass vial and add 5 ml of saturated aqueous sodium bicarbonate to it. 1. Using a Pasteur pipet and rubber bulb, carefully pipet your yellow reaction mixture into this ahco solution. 14. Rinse your old reaction vial with 5 ml of EtOAc and add it to the vial containing the ahco and yellow reaction mixture. 15. Cap the new vial and shake it vigorously. 16. Place the vial down on your benchtop and slowly unscrew the cap to vent any gasses that have built up. 17. Allow the organic layer and the aqueous layer to separate. a. Do you know why many organic solvents and water are not soluble? Based on the densities of the two layers can you predict which is the organic and which is the aqueous? 18. Once the layers have separated, carefully pipet out the aqueous layer and dispense it into another glass vial on your benchtop. You are interested 80

9 Life Sciences 1a Laboratory all 2006 in keeping the organic (yellow) layer in which your product is dissolved but should not discard the aqueous layers you will be removing in case you accidentally mix the layers up. If you do make a mistake, you can always rescue the proper layer from your discard flask. igure. Aqueous extraction of Diolat. A mixture of ethyl acetate and aqueous solvent will be turbid when mixed (left panel). After allowing the mixture to rest, two layers will separate, with the less dense solution above the denser solution (middle panel). Using a glass pipet, extract the aqueous or lower layer (right panel). 19. Wash the organic layer two times (2X) with 5 ml of saturated sodium chloride (acl) solution. Remove the aqueous layer after each washing. a. By Washing we mean add 5 ml of saturated sodium choloride solution to your vial with the yellow reaction mixture. Shake to mix. Allow the layers to separate, and then remove the aqueous (white/clear lower layer) from the vial. 20. Using a scoopula, add a scoop or two of solid anhydrous sodium sulfate (a2so4) to your organic layer. a. Sodium sulfate is a drying reagent and will absorb any residual water sill present after the washings. Hydrated a2so4 is clumpy and hard while dry a2so4 will remain granular. Typically you can use these visual clues to determine when your solution is drywhen granular, non-clumped drying reagent remains in your flask, all of the moisture has been absorbed. However on a scale this small it may be difficult to determine if your a2so4 is clumpy or not, so just swirl the contents of the vial occasionally and allow the drying to take place over approx. 5 minutes. 21. Obtain a fresh glass vial and record the weight of it. 22. Using a pipet, carefully transfer as much of your solution to the fresh vial as possible, while leaving behind the a2so4. 2. Label the vial with your name and T and have your T direct you where to store it until the next lab. The vials will be stored uncapped in a fume 81

10 Life Sciences 1a Laboratory all 2006 hood to allow the solvent to evaporate. During the next lab you will weigh your vial and determine a crude chemical yield of your unpurified DIOLAT. Clean-Up Pour ethyl acetate and hexane into the organic waste blue bucket Pour the aqueous extracts into the aqueous waste blue bucket Place the vial containing a 2 SO 4 into the solid waste bucket Refer to Lab 4-Appendix 1 for instructions on how to clean the glassware that you used Questions (due one week after the completion of the lab) 1. Attach a labeled copy of your results (TLC plate and table describing the amount of product you began with and yield), and describe them. a. What did you do in this experiment? b. On your TLC plate, which spots correspond to Diolat and lexat? c. What are the Rf values for Diolat and lexat? d. Are there any additional spots on your TLC plate? What do you think those correspond to? e. What was your yield in this synthesis reaction? f. Was your yield greater than or less than your theoretical yield? g. If your yield was less than the theoretical yield, explain how this could have occurred. h. If your yield was greater than the theoretical yield, explain how this could have occurred. 2. How many syn-diolat and anti-diolat isoforms are made using our procedure? What compound is used by ovartis to generate syn-diolat? Do you think ovartis generates both syn-diolat isoforms or only one? Why or why not?. In your TLC, the completed reaction product (Diolat) migrated more slowly than the precursor (lexat). What does this indicate about the affinity of Diolat and lexat for the TLC plate? 4. Using TLC, do you think it is possible to distinguish the various isomers of lexat from one another? Do you think you could distinguish the various isomers of Diolat from one another? Explain. 82

11 Life Sciences 1a Laboratory all TLC plates are coated with silica. Silica is a polar substance. If you were to perform a TLC with plates coated with a non-polar substance, how do you think the migration of Diolat and lexat would be altered? Explain. 6. What is the major structural difference between Diolat and luvastatin? Do you think Diolat will bind to HMG-CoA Reductase? Why or why not? 7. luvastatin binds to HMG-CoA Reductase with greater affinity than HMG-CoA. What is the basis for this increased binding affinity? 8. If eukaryotic cells were treated with high doses of fluvastatin, explain how you think this would affect the fluidity of their cell membranes. 8

12 Life Sciences 1a Laboratory all 2006 Appendix 1: The importance of statins In the United States, about 950,000 people die each year of cardiovascular disease. One of the most important risk factors of cardiovascular disease is an elevated cholesterol level. In the United States, over 107 million adults are classified with high or borderline-high cholesterol levels. Cholesterol is ingested with food, and it can also be synthesized. Many people can modify their cholesterol level through simple lifestyle changes such as diet and exercise. Cholesterol levels are also often lowered through the use of a class of pharmaceuticals collectively known as statins. Statins are small-molecules that lower cholesterol levels by inhibiting the body s ability to synthesize cholesterol. Where did statins come from? In the 1950s and 60s, many cholesterol-lowering agents were in clinical use, however all of them had undesirable side-effects or were inappropriate for longterm usage. In the 1960s, cholesterol metabolism was being extensively studied in both humans and animals, and it was discovered that cholesterol in the body could either come from intestinal absorption of dietary cholesterol or through biosynthesis in the body, primarily the liver. It was also revealed that if dietary cholesterol was reduced, biosynthesis increased, and if dietary cholesterol intake increased, biosynthesis decreased. This alteration in biosynthesis is modulated by the activity of HMG-CoA Reductase, an enzyme that converts HMG-CoA into mevalonate, which in turn is used to generate cholesterol. HMG-CoA Reductase Cholesterol The biosynthesis of HMG-CoA to Mevalonate and eventually Cholesterol Thus several groups hypothesized that cholesterol levels could be lowered through the inhibition of de novo biosynthesis, and in the early 1970s several groups began to search for novel compounds that could inhibit the activity of HMG-CoA Reductase. In 1976 two groups published papers describing two HMG- CoA Reductase inhibitors with identical structures isolated from different sources - Akira Endo, Masao Kuroda, and Y. Tsujita identified Mevastatin from a microbe called Penicillium citrinum and Brown et. Al. identified Compactin from Penicillium brevicompactum. 84

13 Life Sciences 1a Laboratory all 2006 HMG-CoA Compactin/Mevastatin (lactone) Compactin/Mevastatin (Acid form) The structure of Compactin/Mevastatin compared to HMG-CoA What do statins look like, and how do they work? HMG-CoA-like Side-chain ester Connecter Dexahydronaphthalene (heteroaromatic) core Compactin/Mevastatin (Acid form) The features of Compactin/Mevastatin Compactin/Mevastatin are characterized by four moieties: 1) the b-hydroxy-dlactone or,5 dihydroxyheptanoic acid (HMG-CoA like), 2) the moiety connecting the lactone and lipophilic group, ) the hexahydronaphthalene core, and 4) the side chain ester. The lactone domain is structurally similar to HMG and binds in the active site of HMG-CoA Reductase. Additionally the hexahydronaphthalene core binds to an adjacent hydrophobic pocket of HMG-CoA Reductase not normally utilized in HMG-CoA substrate binding. Thus with two binding sites, Compactin/Mevastatin binds HMG-CoA Reductase with 10,000 fold higher affinity, and thus is able to out-compete HMG-CoA for binding to HMG-CoA Reductase. 85